Serine proteases belong to the group of proteolytic enzymes that have an intensively studied catalytic mechanism in common. Serine proteases are found in viruses, bacteria and eucaryotes. They include exopeptidases, endopeptidases and oligopeptidases. There are similarities in the reaction mechanism for several of the peptidases with different evolutionary origins. The geometric orientation of the catalytic residues is closely similar, despite the fact that otherwise the protein folds are quite different. A catalytic triade of serine, histidine and aspartate residues in the active site is responsible for efficient hydrolytic cleavage of the peptide bond. Examples of serine proteases include thrombin, factor XIIa, factor IXa, factor Xa, plasmin, tPA, trypsin, chymotrypsin and further proteins like urokinase, tryptase, elastase, kallikrein, complement C, protease A, serine carboxypeptidase II. They are involved in a variety of important processes like, for example, blood coagulation and food digestion. It has been shown that serine protease inhibitors inhibit cellular processes, such as adhesion, migration, free radical production and apoptosis. Intravenously administered serine protease inhibitors provide a protective effect against tissue damage. Small molecule inhibitors have been shown to have a high potential in treatment of different diseases related to hematology, oncology, asthma, inflammation, neurology, pulmonary medicine and immunology. Appropriate serine protease inhibitors may be useful in treatment of disfunctions in the field of thrombotic diseases, asthma, cirrhosis, arthritis, carcinoma, melanoma, restenosis, atheroma, trauma, shock and reperfusion injury.
The investigated enzyme neurotrypsin (WO 98/49322) belongs to the chymotrypsin family, whose members are almost entirely confined to animals. The amino acid sequence of neurotrypsin defines a mosaic protein of 875 amino acids consisting of a Kringle domain, followed by four scavenger receptor cysteine-rich repeats (three in the mouse), and the serine protease domain (FIGS. 1, A and B). Neurotrypsin contains, like thrombin, tPA, trypsin and some other enzymes, an aspartate residue in the bottom of its S1 pocket, therefore showing specificity for basic amino acids at this binding site. The structural similarity of neurotrypsin to the proteases of the blood coagulation cascade and the fibrinolytic system, such as factor X, factor IX, thrombin, tissue plasminogen activator, and plasmin suggests that it may be an element of a protease-driven extracellular signaling mechanism in the nervous system. (Gschwend, T. P., et al., Molec. Cell Neurosci. 9: 207-219, 1997; Proba, K., et al., Biochim. Biophys. Acta 1396: 143-147, 1998).
As will be shown hereinbelow, neurotrypsin is located at the presynaptic nerve terminal of synapses of the central nervous system (CNS) and at the neuromuscular junction (NMJ). The synapse is the connection between nerve cells (neurons) where messages are communicated in the form of chemical substances, termed neurotransmitters. The synapse is composed of a presynaptic terminal formed by the signal-emitting cell and the postsynaptic specialization of the signal-receiving cell. Neurotransmitters released from the presynaptic terminal cross the synaptic cleft and bind to the neurotransmitter receptors in the postsynaptic specialization. Upon binding of the neurotransmitter the receptor induces the generation of an electrical pulse in the postsynaptic cell. Signal transmission between two neurons is the basis of neuronal function. Brain functions are the result of the specific assembly of an enormous number of neurons to information-processing networks.
The majority of synapses is found in the central nervous system (CNS, brain), where every synapse connects two neurons. By such bilateral point-to-point connections, every neuron may connect to thousands of other neurons. However, synapses also connect a neuron to a gland or a muscle cell. The neuromuscular junction (NMJ, muscle end-plate) is the synapse that connects a nerve cell with a striated muscle cell. Synapses located outside of the brain, the brain stem and the spinal cord are termed peripheral nervous system (PNS) synapses. CNS synapses and PNS synapses exhibit many structural and functional commonalities and share many of their molecular components (synaptic molecules). Therefore, synaptic target molecules may be useful for targeting synaptic functions of both the CNS and the PNS.
Skeletal muscle atrophy (sarcopenia), defined as the loss of muscle mass and strength, plays a major role in the pathogenesis of frailty and functional impairment that occurs with old age. It plays a major role in the loss of muscular strength, decreased metabolic rate, gradual reduction of bone density and decreased aerobic capacity (Doherty, T. J., J. Appl. Physiol. 95: 1717-1727, 2003). The loss of muscle mass manifests as a decrease in the cross-sectional area of the muscle with age, which has been determined to result from a combined effect of a reduction in both the number of muscle fibers and the thickness of the individual remaining fibers.
Over the past years, considerable progress has been made in the identification and characterization of factors contributing to the degradation of muscle mass. Important genes associated with these processes encode ubiquitin protein ligases that were found increased in atrophic muscle. Among the factors that have a hypertrophic activity and, as such, block atrophy, insulin-like growth factor 1 (IGF-1) has been found to play an essential role. This and several other regulatory pathways controlling skeletal muscle mass have been investigated intensively (for a review see: Glass, D. J., Nature Cell Biol. 5: 87-90, 2003). In spite of important progress in both the characterization of the molecular mechanisms that control muscle degradation leading to atrophy and the hypertrophic effects of insulin-like growth factor, and in spite of the fact that several companies work on the development of drugs capable of stimulating the increase of muscle mass, no drugs have been approved up to now.
A morphological hallmark of the skeletal muscle atrophy found at old age (sarcopenia) is a considerable reduction of the number of muscle fibers. Ample evidence from numerous independent studies supports that neural input to a fraction of the muscle fibers is disrupted with age, resulting in subsequent atrophy and eventually the disappearance of the denervated fibers (Kamal, H. K., Nutrition Reviews 61: 157-167, 2003). Another characteristic feature of the skeletal muscle atrophy found at old age is a coincidence of the muscular atrophy with a considerable reduction of the number of motoneurons (Welle, S., Can. J. Appl. Physiol. 27: 19-41, 2002) and a marked structural alteration of the neuromuscular junction (Tapia, J. C. et al., Abstract Viewer/Itinerary Planner, Washington D.C.: Society for Neuroscience). These characteristics indicate that a significant age-related deterioration of the structure and the function of the neuromuscular junction is a major contributing factor to a process that ultimately results in a structural and functional denervation. Denervated muscle fibers that do not receive compensatory reinnervation within weeks become progressively atrophic and eventually disappear.
Schizophrenia is a chronic, severe, and disabling brain disease. Approximately 1% of the world population develops schizophrenia during their lifetime. Individuals who develop schizophrenia experience severe suffering. Approximately 10% commit suicide. Although schizophrenia affects men and women with equal frequency, the disorder often appears earlier in men, usually in the late teens or early twenties, than in women, who are generally affected in the twenties to early thirties. People with schizophrenia often suffer terrifying symptoms such as hearing internal voices not heard by others, or believing that other people are reading their minds, controlling their thoughts, or plotting to harm them. These symptoms may leave them fearful and withdrawn. Their speech and behavior can be so disorganized that they may be incomprehensible or frightening to others. The currently available treatments of schizophrenia reduce suffering considerably, but approximately ⅔ of the people affected by schizophrenia require public assistance within a few years after onset. The majority of them are unable to return to work or school and have relatively little or no social interactions, and most people with schizophrenia continue to suffer some symptoms throughout their lives. It has been estimated that no more than one in five individuals recovers completely. Therefore schizophrenia is one of the most important public health problems world-wide, and the costs to society are counted in billions of dollars.
The currently most consistent neuropathological finding in brains of schizophrenic patients is a reduction of the number of synapses in the gray matter of the central nervous system, which is reflected by a decrease in the volume of the neuropil (the synaptic area). No evidence for neuronal degeneration is observed. Typically, the number of neurons counted per area of tissue is rather increased, an observation explained by a selective decrease in the number of synapses in the neuropil area between the neurons while the number of neuronal cell somas remained constant. The phenomenon has been reported over the past two decades by several independent studies on post mortem material and has been found most extensive in the prefrontal cortex. The literature documenting this observation has been carefully reviewed by Selemon, L. D. and Goldman-Rakic, P. S. (Psychiatry 45: 17-25, 1999). McGlashan, T. H. and Hoffman, R. E. (Arch. Gen. Psychiatry 57: 637-648, 2000) summarized the essential morphological, developmental, electrophysiological, and metabolic observations in schizophrenia in the light of the “excessive synaptic pruning” hypothesis and came to the conclusion that “excessive synaptic pruning” or “developmentally reduced synaptic connectivity” is an increasingly attractive pathophysiological model of schizophrenia. Based on this model, schizophrenia arises from critically reduced synaptic connectedness as a result of developmental disturbances of synaptogenesis during gestation and early childhood and/or excessive synaptic pruning during adolescence. The model accounts for the phenomenology of the disorder, the symptomatic states, the onset, neurodevelopmental deficits, window of deterioration, sex differences in clinical presentation, course determined by age of onset, and preservation of the schizophrenic genotype in the population despite diminished phenotypic fecundity.
Cognitive enhancers are drugs aimed at preventing, improving, or treating cognitive deficits at both the clinical and subclinical level. Such drugs are beneficial for the treatment of memory difficulties of elderly people who have not progressed to Alzheimer's disease (mild cognitive impairment). However, such drugs are also beneficial for the improvement of cognitive functions in patients with the established diagnosis of Alzheimer's disease or other diseases associated with dementia or for the improvement of cognitive functions in posttraumatic cognitive dysfunction, as well as for the improvement of the age-related impairment of cognitive functions that are considered as a normal feature of the ageing process.
Mild cognitive impairment is a widely cited concept in clinical research on ageing-related cognitive disorders (Ritchie, K. and Touchon, J., The Lancet 355: 225-228, 2000). It refers generally to subclinical complaints of memory functioning in elderly people, which are judged to have a high probability of evolving towards Alzheimer's disease. The identification of people at potential risk for dementia with a view to early therapeutic intervention is important, because it may lessen distress for both patient and family, minimize the risk of accidents, prolong autonomy, and perhaps even ultimately prevent the onset of the process leading to dementia itself.
The impairment of cognitive functions without dementia is so common among elderly people that it is considered by many as an inevitable feature of the ageing process. Nonetheless, it has acquired clinical significance because of the difficulties patients may have with carrying out everyday activities. Although the range of impairments seen in populations without dementia is extremely broad, several clinical labels have been proposed to describe this tail-end of the normal cognitive range. One of the earliest was benign senescent forgetfulness. Its clinical features include an inability to recall minor detail, the forgetting of remote as opposed to recent events, and awareness of memory problems. The term ageing-associated cognitive decline refers to a wider range of cognitive functions (attention, memory, learning, thinking, language, and visuospatial function), and is diagnosed by reference to norms for elderly people. Prescription of cognitive enhancers may prolong the capacity of the affected individuals to carry out their daily activities and, thus, prolong their autonomy. Other disorders associated at least in part of the affected individuals with cognitive impairments that may eventually lead to dementia include Parkinson's disease, multiple sclerosis, stroke, and head trauma. The prescription of cognitive enhancer drugs may also improve cognitive functions in these patients.